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Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes▿

机译:检测肺炎克雷伯氏菌碳青霉烯酶基因的实时PCR方法的开发和评估

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摘要

We developed a novel real-time PCR assay to detect Klebsiella pneumoniae carbapenemases (KPCs) and used this assay to screen clinical isolates of K. pneumoniae and Klebsiella oxytoca for the presence of blaKPC genes. The TaqMan real-time PCR assay amplified a 399-bp product from the blaKPC gene. The amplicon was designed so that the genes for isoenzymes KPC-1, -2, and -3 could be easily distinguished by subsequent restriction digestion of the amplicon with the enzymes BstNI and RsaI. The assay was validated with reference strains obtained from the Centers for Disease Control and Prevention that contained each of the three described isoenzymes and 69 extended-spectrum β-lactamase-producing clinical isolates (39 K. pneumoniae and 30 K. oxytoca isolates). Subsequently, the blaKPC PCR assay was used to confirm the presence of blaKPC genes in any meropenem-resistant Klebsiella spp. The PCR assay detected blaKPC in all of the reference strains, in 6 of 7 meropenem-resistant isolates, and in 0 of 62 meropenem-susceptible clinical isolates. The PCR assay was then used to confirm the presence of blaKPC in an additional 20 meropenem-resistant isolates from 16 patients. Restriction digestion of the PCR amplicons identified two blaKPC gene variants in our patient population: 9 isolates with C and 17 with T at nucleotide 944, consistent with blaKPC-2 and blaKPC-3, respectively. The real-time PCR assay is a rapid and accurate method to detect all KPC isoenzymes and was useful in documenting the presence and dissemination of KPC-producing strains in our patient population.
机译:我们开发了一种新颖的实时PCR检测试剂盒,以检测肺炎克雷伯菌肺炎克雷伯菌(KPC),并使用此测定法筛选肺炎克雷伯菌和产氧克雷伯菌的临床分离株是否存在blaKPC基因。 TaqMan实时PCR分析扩增了blaKPC基因的399 bp产物。对扩增子进行设计,以便通过随后用BstNI和RsaI酶对扩增子进行限制性酶切消化,可以轻松地区分同工酶KPC-1,-2和-3的基因。用从疾病控制和预防中心获得的参考菌株对测定进行验证,该参考菌株包含上述三种同功酶和69种产生广谱β-内酰胺酶的临床分离株(39 K.肺炎克雷伯菌和30 K. oxytoca分离株)。随后,使用blaKPC PCR分析法确认任何耐美罗培南的克雷伯菌属中存在blaKPC基因。 PCR测定法在所有参考菌株中,在7个耐美罗培南的分离株中的6个和62个对美洛培南敏感的临床分离株中的0个中检测到blaKPC。然后使用PCR测定法来确认blaKPC是否存在于来自16例患者的另外20株耐美罗培南的分离株中。 PCR扩增子的限制性酶切鉴定在我们的患者群体中鉴定出两个blaKPC基因变异:9个C分离株和17个T核苷酸为944的分离株,分别与blaKPC-2和blaKPC-3一致。实时PCR分析是一种快速准确的检测所有KPC同工酶的方法,可用于记录我们患者人群中KPC产生菌株的存在和传播。

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